help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ricken*, A.
Right arrow Articles by Farookhi, R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ricken*, A.
Right arrow Articles by Farookhi, R.
Endocrinology Vol. 143, No. 7 2741-2749
Copyright © 2002 by The Endocrine Society

REPRODUCTION-DEVELOPMENT

Wnt Signaling in the Ovary: Identification and Compartmentalized Expression of wnt-2, wnt-2b, and Frizzled-4 mRNAs

Albert Ricken*1, Paul Lochhead, Maria Kontogiannea and Riaz Farookhi

Department of Obstetrics and Gynecology (A.R., P.L., M.K., R.F.) and Department of Physiology (R.F.), McGill University, Montréal, Québec, Canada H3A 1A1

Address all correspondence and requests for reprints to: Riaz Farookhi, F3.44 Royal Victoria Hospital, 687 Pine Avenue West, Montréal, Québec, Canada H3A 1A1. E-mail: . Riaz.Farookhi{at}MUHC.McGill.ca


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Ovarian cadherins, in addition to acting as structural (adhesion) molecules, also function as modulators of gene activity. The dual role of ß-catenin as an intracellular component of the cadherin adhesion complex and as a transcription factor provides a possible explanation for these cadherin effects. Because the transcriptional activity of ß-catenin is dependent on activation by the wnt signaling cascade, we examined whether components of this cascade are expressed in the rat ovary.

Using RT-PCR with degenerate primers on RNA from ovaries of hormone-stimulated immature rats, we identified transcripts for wnt-2 and wnt-2b. RT-PCR and in situ hybridization (ISH) demonstrated that granulosa cells express wnt-2 mRNA. Because the sequence for rat wnt-2b has not been reported, we obtained additional sequence by screening a rat ovarian cDNA library. RT-PCR analysis, using primers designed from this wnt-2b cDNA sequence, failed to detect transcripts in the ovarian follicular compartment (granulosa and oocyte). ISH revealed that the ovarian surface epithelium expresses wnt-2b mRNA. Using a similar degenerate RT-PCR approach, we detected expression of a putative wnt receptor, frizzled-4 (fzd-4), and a cytoplasmic component of the wnt signaling cascade, disheveled-2 (dsh-2), in the rat ovary. Further analyses using both RT-PCR and ISH indicated that granulosa cells express fzd-4 mRNA.

The expression of wnt-2b transcripts in rat ovarian surface epithelium prompted us to examine whether the homologous gene is expressed in human ovarian cancer cell lines. RT-PCR, using degenerate and specific primers for wnts, on RNA from five ovarian cancer cell lines confirmed the expression of transcripts for wnt-2b. Two additional wnt transcripts (wnt-5a and wnt-11) were detected in the cancer cell lines and in the rat ovary.

These results demonstrate that transcripts corresponding to components of the wnt signaling cascade are expressed in the immature rat ovary. The localization of these transcripts in specific ovarian compartments suggests that this signal transduction pathway may be involved in follicular development and ovarian function. Furthermore, because wnts have been implicated in the oncogenic transformation of epithelial cells, our results raise the possibility that aberrant wnt expression may be involved in ovarian tumorigenesis in humans.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE CADHERINS COMPRISE a family of structurally related transmembrane glycoproteins that mediate calcium-dependent cell adhesion (1, 2, 3). Previous studies have shown that cadherins play an important role in ovarian development and function (reviewed in Ref. 3). Besides structural activities (4, 5, 6, 7, 8), ovarian cadherins are involved in the modulation of gonadotropin-stimulated signal transduction (9), granulosa cell differentiation (10, 11, 12), and ovarian cell survival (13, 14, 15). The latter observations indicate that, in addition to their role as cell scaffolding molecules, ovarian cadherins are involved in signal transduction leading to the modulation of gene expression. A series of recent genetic and biochemical studies in Drosophila, Caenorhobditis elegans, Xenopus and mouse have revealed that an intracellular component of the cadherin adhesion complex, ß-catenin, is also a component of the wnt signaling pathway (reviewed in Refs. 16 and 17). Cellular ß-catenin is usually sequestered at the plasma membrane in the cadherin adhesion complex. In the absence of wnt signaling, nonsequestered cytoplasmic ß-catenin is phosphorylated, which allows its ubiquination and subsequent destruction (16, 17). Wnt signaling prevents ß-catenin phosphorylation, allowing cytoplasmic ß-catenin to function as a transcription factor.

Wnt genes encode a family of secreted short-range signaling glycoproteins (16, 17). In mammals almost twenty different wnts have been described (the interested reader is directed to the Wnt gene page at http://www.stanford.edu/~rnusse/wntwindow.html). Wnt target cells express members of the frizzled (fzd) receptor family that are serpentine, seven transmembrane spanning, potential G protein-coupled receptors (17, 18). Wnt binding to fzd activates at least two distinct intracellular signaling pathways. One, termed the canonical wnt signaling pathway, leads to the accumulation/stabilization of cytosolic ("free") ß-catenin. The cytosolic ß-catenin serves, after translocation to the cell nucleus, as a partner for members of the T cell factor/lymphocyte enhancing factor family of transcription factors resulting in the modulation of target gene activity. The other pathway, which also involves wnt binding to fzd, promotes an increase in intracellular calcium concentration and activation of calmodulin-dependent kinase II or protein kinase C as a means of altering target cell function (19). The type of fzd receptor (presently, ten mammalian fzds have been identified) appears to be the determinant of which pathway is activated (20). There is evidence, however, that the relative affinity of fzds for different wnts is involved in pathway selection (21). These observations indicate that the choice of signaling pathways can reside in the ligand, the receptor, or both.

The possibility that the effects observed in ovarian cells, as a consequence of cadherin modulation (9, 10, 11, 12, 13, 14, 15) and ß-catenin release, could affect wnt signaling prompted us to examine whether components of the wnt signaling cascade are expressed in the ovary. Previous studies (22) have demonstrated a critical role for wnt-4 in embryonic ovarian development. Wnt-7a has been shown to effect sex-specific differentiation of the reproductive tract (23). To our knowledge, however, the potential for wnt signaling in the developed ovary has not been addressed. Here, we report that components of the wnt signaling pathway are expressed in the immature rat ovary, and that their expression is localized to specific ovarian compartments.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Immature (21 d old) female Sprague Dawley rats were purchased from Charles River Canada (St. Constant, Québec, Canada). Animals were housed in a centralized animal facility under constant environmental conditions and had free access to rat chow and water. All animal treatments and procedures were approved by the Animal Care Committee of the Royal Victoria Hospital and complied with the regulations established by the Canadian Council for Animal Care.

Gonadotropin-treated rats were used in the studies involving the PCR-based screening for expression of wnt signaling components. Rats were injected sc at 22 d of age with 20 IU of equine chorionic gonadotropin (CG) (eCG; Equinex, Ayerst, Montréal, Québec, Canada) followed 48 h later by an injection (sc) of 10 IU human CG (hCG, APL, Ayerst). At 48 h after eCG, or at 24 h or 8 d after the human CG treatment, groups of rats were killed, the ovaries removed, cleared of adherent connective tissue, and frozen in liquid nitrogen. Ovaries were stored at -80 C until the time of RNA extraction.

Germinal vesicle intact and MII oocytes were isolated from untreated 22- to 24-d-old animals and eCG-hCG superovulated rats, respectively. Oocytes, stripped of adherent cumulus cells, were isolated following the procedures of McClay and Clarke (24). Follicular granulosa cells were isolated from eCG-treated ovaries as described previously (25).

RNA extraction
Ovaries.
Total RNA was extracted from pulverized frozen ovaries using the guanidium thiocyanate-phenol-chloroform method (26). RNA yield was assessed by spectrophotometry, and RNA integrity confirmed by agarose gel electrophoresis.

Oocytes and granulosa cells.
Total RNA was recovered from these cells using Trizol (Life Technologies, Inc., Burlington, Ontario, Canada) as per the manufacturer’s instructions. Oocytes (pools of 30–50 GV intact or 15–40 MII) or granulosa cells were collected in a total volume of 100 µl Trizol. Glycogen (10 µg) was added before alcohol precipitation of the RNA extracts. The oocyte RNA was reconstituted in sterile water at a concentration corresponding to the equivalent of 5 oocytes/1 µl of water. RNA from granulosa cell preparations was reconstituted in 10 µl of water.

RT-PCR
The expression of wnt signaling components in ovaries was assessed by RT-PCR using degenerate primers. The degenerate oligonucleotides corresponded to conserved amino acid motifs in wnt (27, 28), the putative wnt receptor fzd (29, 30), and disheveled (dsh) (31) gene families. The oligonucleotides were synthesized at the Sheldon Biotechnology Center (McGill University). The sequences for the conserved amino acid motifs and the corresponding primer sets are listed in Table 1Go.


View this table:
[in this window]
[in a new window]
  		Table 1. Degenerate primer sequences for wnt, fzd, and dsh

 
One microgram of RNA was reverse-transcribed using Moloney murine leukemia virus (200 U, Life Technologies, Inc.) and each one of the listed downstream primers (Table 1Go). Subsequent PCR was performed using the individual primers sets listed in Table 1Go. In all cases the same amplification conditions were used; these consisted of an initial denaturation step for 5 min at 95 C followed by 35 cycles of 95 C for 60 sec, 55 C for 1.5 min, 72 C for 1 min with a final extension at 72 C for 5 min. The amplified products were cloned into pCR2.1 using the TA cloning kit (Invitrogen, Burlington, Ontario, Canada). Clones harboring cDNA inserts of the expected size (350–400 bp for wnt; 210–340 bp for fzd; 280–300 bp for dsh) were isolated and sequenced. Subsequent identification of the cDNA inserts was obtained by interrogation of GenBank (BLAST search).

The expression of specific wnts and wnt signaling components in granulosa cells and oocytes was examined by RT-PCR. RNA, from these isolated cell types (see above) was reverse transcribed using random hexamers (Amersham Pharmacia Biotech, Montréal, Québec, Canada) and Superscript II (Life Technologies, Inc.). PCR was performed using the conditions described above and oligonucleotide primers corresponding to consensus sequences from the published rat and mouse wnt-2 (32), wnt-2b (33), and fzd-4 (29) sequences, respectively. The upstream and downstream primers for wnt-2 were 5'-TGGACAGCTGCGAAGTTATG-3' and 5'-AACAACCCAGAGGTCCAGTG-3', respectively; for wnt-2b, 5'-GATTCCTGAAGCTGGAGTGC-3' and 5'-AGTTGTGTCATAC CCTCGGC-3'; and for fzd-4, 5'-GCCAATGTGCACAGAGAAGA-3' and 5'-AGGCAAACCC AAATTCTCTCA-3'. In addition, PCR using primers for hypoxanthine phosphoribosyltransferase (HPRT), a housekeeping gene (34), rat FSH receptor (35) and ZP3 (36) were used as controls for RNA integrity and assessment of the relative purity of the tissue fractions. The upstream and downstream primers corresponded to nucleotides (nt) 329–349 and 656–680 for HPRT (GenBank accession no. M63983), nt 127–148 and 633–652 for rat FSH receptor (L02842) and nt 539–558 and 1095–1115 for ZP3 (D78482). Because the products generated using these primers span an intron-exon boundary in the gene, genomic contamination of the RNA preparations would be detected.

cDNA library screening
A number of clones, generated using the degenerate RT-PCR strategy, contained the sequence corresponding to human and mouse wnt-2b. Because the corresponding rat ortholog has not been described, we screened a rat ovarian cDNA library (generously provided by Dr. Michael Melner, Vanderbilt University Medical Center, Nashville, TN) using PCR with specific wnt-2b primers. The upstream and downstream primers were derived from the published mouse wnt-2b sequence (33) and were, respectively, 5'-GATTCCTGAAGCTGGAGTGC-3' and 5'-AGTTGT GTCATACCCTCGGC-3'. The library was screened by successive fractionation following the procedures of Bockmann et al. (37). Phage DNA from the single plaque corresponding to the wnt-2b insert was isolated and amplified using T3 and T7 primers. The 1.7-kb insert was cloned into pCR2.1 (TA cloning kit, Invitrogen) and confirmed as the rat wnt2b ortholog by sequencing.

In situ hybridization (ISH)
Tissue preparation.
Ovaries from untreated immature rats were fixed in 4% paraformaldehyde in PBS, dehydrated, and embedded in paraffin. Ten-micrometer sections were cut, mounted on poly-L-lysine-coated slides, deparaffinized in xylene, and rehydrated through graded ethanol. Hydrated sections were immersed in 0.2 N HCl for 20 min, permeabilized in 0.3% Triton X-100 and treated with proteinase K (10 µg/ml) for 30 min at 37 C. Sections were postfixed with 4% paraformaldehyde, acetylated, and incubated in prehybridization solution at 37 C for 1 h. Hybridization to digoxygenin (DIG)-labeled probes was conducted at 37 C overnight in 10 mM Tris buffer (pH 7.5) containing 50% formamide, 2x saline sodium citrate (SSC), 0.5% sodium dodecyl sulfate, 12.5x Denhardt’s solution, 10% dextran sulfate, and 250 µg/ml denatured salmon sperm DNA. The slides were washed at 37 C (2 x 15 min with 2x SSC, 2 x 15 min with 1x SSC, and 2 x 15 min with 0.25x SSC). Hybridized DIG-probes were detected using anti-DIG antibody conjugated to alkaline phosphatase, and visualized using a nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate solution (Roche Diagnostics, Laval, Québec, Canada).

Probe preparation
Single-stranded DIG-labeled DNA probes for wnt-2b were prepared from the appropriate template according to the procedures described by Suter and Steward (38). Briefly, the procedure involves the generation of short sense or antisense fragments by primer extension. Primers were designed to correspond to specific sequences in the template, generally separated from one another by 300–400 bp. By controlling the extension period, this approach generates multiple DNA fragments suitable for ISH. The labeled probes were precipitated, resuspended, sonicated, and boiled for 60 min in 300 µl of hybridization buffer.

DIG-labeled DNA probes for wnt-2 and fzd-4 failed to provide hybridization in ovary sections. We suspect that this could reflect the low abundance of these mRNAs in granulosa cells. Hence, DIG-labeled sense and antisense riboprobes for wnt-2 and fzd-4 were prepared by in vitro transcription using T7 or SP6 RNA polymerase, respectively. The entire wnt-2 and fzd-4 cDNA sequences in pCR2.1 were excised using XbaI and SacI and ligated within the corresponding restriction sites of pGEM-3Z (Promega Corp., Madison, WI). Correct orientation of the cDNA inserts was verified by sequencing.

Ovarian cancer cell lines
Our ISH studies on rat ovaries indicated that wnt-2b was expressed, almost exclusively, in the ovarian surface epithelium (OSE) (see Results). Because the overwhelming majority of ovarian cancers in humans arise from this tissue (39), we examined human OSE cancer cell lines for the expression of wnt-2b and other wnts.

Five human ovarian cancer lines are maintained in our laboratory. Four of them (SKOV-3, CaOV3, OVCAR3, and SW626) were obtained from ATCC (Manassas, VA). The fifth, termed Hey, was a gift from Dr. J. Dorrington (Banting and Best Institute, University of Toronto, Toronto, Canada). The provenance and characteristics of this cell line have been reported (40). All cell lines, except OVCAR-3, were maintained in MEM, Life Technologies, Inc., Burlington, Ontario, Canada) supplemented with 10% fetal calf serum, and fortified with 2 mM L-glutamine and 0.1 mM nonessential amino acids (Life Technologies, Inc.). OVCAR-3 cells were maintained in fortified RPMI 1640 containing 20% fetal calf serum (Life Technologies, Inc.).

Total RNA was prepared from the cell lines by direct dissolution of the culture monolayers in solution D [4 M guanidinium thiocyanate, 25 mM sodium citrate, pH 7, 0.5% sarcosyl, 0.1 M ß-mercaptoethanol (26)]. RNA was recovered after phenol-chloroform extraction and alcohol precipitation.

The degenerate RT-PCR strategy, outlined above, was used with the RNA prepared from the cell lines. Amplified fragments were cloned into pCR2.1. Clones containing appropriate sized inserts were isolated and sequenced. The expression of the particular wnts identified (wnt-2b, wnt-5a, wnt-11) was confirmed by RT-PCR using specific primers derived from the reported GenBank sequences.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
mRNAs for Wnt signaling components are expressed in the rat ovary
Our RT-PCR strategy, using degenerate primers for wnt, fzd, and dsh yielded appropriately sized products from rat ovarian RNA. The PCR products were ligated into pCR2.1, and a number of independent clones (at least twenty clones for each product) were isolated and sequenced. Six independent clones, generated using the degenerate wnt primers, showed high sequence homology to mouse (33) and human wnt-2b cDNA (41, 42). Two additional clones were found to contain sequences that were identical to rat wnt-2 cDNA (32). Four clones generated using the degenerate fzd primers were identical in sequence to rat fzd-4 (GenBank accession no. AF183910). Only one clone yielded a product showing homology to mouse dsh-2 (43). Although these results were obtained using total ovarian RNA isolated from eCG-hCG-treated rats, similar results were observed using ovarian RNA from animals treated with eCG only. These data indicate that the rat ovary expresses transcripts for at least two wnts, one fzd, and one dsh.

Cloning and isolation of additional sequence for rat wnt2b
The sequence for rat wnt-2b has not been reported. This sequence is likely to be of interest because differences between the N-terminal amino acid sequence of the published mouse (33) and human (41, 42) wnt-2b sequences have been reported. We, therefore, attempted to obtain additional sequence information for rat ovarian wnt-2b. We screened a rat ovarian cDNA library for wnt-2b. A partial rat wnt-2b cDNA sequence (Fig. 1Go; deposited as GenBank accession no. AF20487) was isolated.



View larger version (74K):
[in this window]
[in a new window]
  		Figure 1. Sequence of the cloned rat wnt-2b cDNA fragment. Because the cloned sequence began within the ORF, nucleotide numbering was started arbritarily at the first base sequenced. The underlined bases represent the stop codon in the longest reading frame. The sequence has been deposited in GenBank under accession no. AF204873.

 
The isolated sequence comprised 1540 bp and contained an open reading frame (ORF) of 936 bp (Fig. 1Go). The 3' untranslated region of rat wnt-2b is 604 bp and contains a polyadenylation site. The partial wnt-2b ORF encodes 311 amino acid residues, contains two potential N-glycosylation sites at positions 115 and 281, and the proper occurrence of 23 of the 24 cysteine residues, which are highly conserved among members of the wnt family (Fig. 2Go). Comparison of the amino acid sequence to that of the mouse and human protein show them to be almost completely identical (>99% similarity; Fig. 2Go). Homology of the rat wnt-2b ORF, at the nucleotide level, with the human and mouse cDNA sequence is 95% and 89%, respectively (not shown).



View larger version (33K):
[in this window]
[in a new window]
  		Figure 2. Comparison of the deduced rat, mouse (33 ), and human (41 ) wnt-2b amino acid sequences. Only the regions of the mouse and human sequence corresponding to the cloned rat coding fragment are shown. Conserved cysteine residues (bold) and potential N-glycosylation sites (underlined) are indicated.

 
Wnt-2, wnt-2b, and fzd-4 transcripts are expressed in discrete compartments of the ovary
RT-PCR was carried out on RNA extracted from isolated granulosa cells, and oocytes from eCG-primed rat ovaries. Specific primers corresponding to the sequences of rat wnt-2, wnt-2b, and fzd-4 cDNAs were used (Fig. 3Go). No products were obtained using the wnt-2b primers with RNA from granulosa cells or oocytes, although RNA from whole ovary as well as lung, a tissue known to express both wnt-2b (33) and wnt-2 (32), were positive. In contrast, wnt-2 and fzd-4 amplicons were observed for the isolated granulosa cells. RT-PCRs with cell type-specific primers, e.g. for ZP3 (oocytes) and FSH receptor (granulosa cells), as well as for the housekeeping enzyme HPRT as an internal standard (34), gave the expected results (results for HPRT only are shown in Fig. 3Go). These data demonstrate a degree of compartmentalization of the wnt signaling components within the ovary. Wnt-2 and fzd-4 mRNAs are expressed within the follicle, specifically in granulosa cells. Wnt-2b transcripts, although detected in the whole ovary, are not expressed in either the membrana granulosa or oocyte. To gain insight into which ovarian compartments might express wnt-2b, its ovarian expression was examined using ISH.



View larger version (35K):
[in this window]
[in a new window]
  		Figure 3. Representative RT-PCR analyses of wnt signaling components expressed in rat oocytes, granulosa cells, ovary and lung (positive control). PCR products, derived from amplification with specific primers for wnt-2 (top panel), wnt-2b (second panel), fzd-4 (third panel) and the housekeeping gene HPRT (bottom panel), are shown. Granuosa cells, but not oocytes, express wnt-2 and fzd-4. Neither granulosa cells nor oocytes express wnt-2b although wnt-2b is expressed in the whole ovary. Amplified products were resolved on 1.5% agarose gels and visualized by eithidium bromide staining. Size markers (bp) are shown in the left lanes. Similar analyses with at least two different RNA preparations from the same tissues provided the same results.

 
Figure 4Go illustrates the results from a representative ISH on sections of rat ovary. Hybridization of wnt-2b antisense probes occurred over the surface epithelium (Fig. 4AGo). The hybridization reaction was judged as even around the entire surface epithelium preserved in the sections. In particular, no differences were noted between parts of the surface epithelium covering regions with nongrowing (primordial) follicles or follicles at early developmental stages (preantral follicles) and those covering more mature follicles (antral follicles). Except for hybridization of the wnt-2b antisense probes over structures that appeared to correspond to the rete ovarii or displaced surface epithelium (Fig. 4CGo), no other specific hybridization was discernable. In accordance with the RT-PCR data, no hybridization for wnt-2b was observed for the membrana granulosa or oocytes. Hybridization of sense wnt-2b probes, in all cases, resulted in only background levels of staining (Fig. 4Go, B and D). These results attest to the predominant compartmentalization of wnt-2b mRNA expression in the rat ovarian surface epithelium.



View larger version (109K):
[in this window]
[in a new window]
  		Figure 4. Representative ISH of rat ovary sections with digoxygenin-labeled wnt-2b DNA probes. Hybridization with the antisense probe revealed wnt-2b expression confined to the ovarian surface epithelium (A, arrow) and to epithelial structures within the ovary (C, asterisk). Panels B and D illustrate the absence of hybridization using the sense probe on adjacent sections (magnification, x100). Sections from at least three to four different animals were examined and provided the same result.

 
Wnt-2 ISH of ovarian sections is shown in Fig. 5Go. In agreement with the RT-PCR results, described above, wnt-2 mRNA appears to be expressed exclusively in granulosa cells. Positive staining was observed in granulosa cells of follicles at all stages of development (Fig. 5AGo), but appears to be more pronounced in large preantral and antral follicles (Fig. 5Go, B and D). Although staining is evident throughout the entire membrana granulosa of larger follicles, one has the impression notably in the large antral follicles that the staining is more intense in the cumulus and more centrally located granulosa cells (Fig. 5Go, B and C). No staining was seen in theca-interstitial cells or in the ovarian surface epithelium. Hybridization with the wnt-2 sense probe revealed only background levels of staining.



View larger version (118K):
[in this window]
[in a new window]
  		Figure 5. Representative ISH of ovary sections with antisense (A–D) or sense (E) digoxygenin-labeled wnt-2 riboprobes. A and B, Images (x150 and x125, respectively) of ovary sections highlighting follicles at various stages of development. Panels C and D illustrate higher magnification (x250 and x300) images. Note the higher intensity of staining in the cumulus/peri-oocyte granulosa cells (arrowheads) compared with the mural cells (arrow). This is more apparent in the follicle shown in C. The oocytes and surface epithelium reveal either no or low levels of labeling. Panel E illustrates hybridization with the sense riboprobe. O, oocyte; g, granulosa cells; se, surface epithelium. Random sections from the ovaries of at least three to four other animals provided the same results.

 
Figure 6Go illustrates the results from ISH using antisense probes corresponding to fzd-4. As was the case for wnt-2, only the membrana granulosa of growing follicles revealed fzd-4 mRNA expression. Unlike wnt-2, however, staining for fzd-4 was even throughout the membrana granulosa. Background levels of staining are seen after hybridization with the sense probe. The results for wnt-2 and fzd-4 mRNA expression indicate that cells of the membrana granulosa are both the source of, and potential targets for wnt.



View larger version (95K):
[in this window]
[in a new window]
  		Figure 6. Representative ISH of ovary sections with antisense (A–C) or sense (D) digoxygenin-labeled fzd-4 riboprobes. Note the even distribution of staining throughout the membrana granulosa of all follicles. A and B, Images (both at x150) of ovary sections highlighting follicles at various stages of development. Panel C illustrates a higher magnification (x250) image. Panel D illustrates hybridization with the sense riboprobe. O, Oocyte; g, granulosa cells; se, surface epithelium. Random sections from the ovaries of at least three to four other animals provided the same results.

 
Wnt-2b mRNA is expressed in human ovarian cancer cells
The overwhelming majority of ovarian cancers arise from the surface epithelium (39). The expression of wnt-2b in the rat ovarian surface epithelium prompted us to examine whether or not this wnt and additional wnts are expressed in human ovarian cancer cells. Five ovarian cancer cell lines (SKOV-3, HEY, CAOV-3, OVCAR, SW626) were tested. The same RT-PCR approach, using degenerate primers was used, followed by confirmation with specific primers. We identified mRNAs for three wnts, wnt-2b, wnt-5a, and wnt-11, in all of these ovarian cancer cell lines (Fig. 7Go). No amplification products were detected when specific primers for human wnt-2 were used on RNA from these cell lines (data not shown). Testing of rat ovarian RNA with primers specific for wnt-5a and wnt-11 yielded amplification products for both of these wnts (data not shown). These observations indicate that wnt-2b is expressed by human ovarian surface epithelial cells along with other wnts.



View larger version (45K):
[in this window]
[in a new window]
  		Figure 7. Representative RT-PCR analyses of wnt expression in human ovarian cancer cell lines. Specific primers corresponding to wnt-2b, wnt-5a and wnt-11 were used. The three ovarian cancer lines depicted here (SKOV3 OVCAR and CAOV-3) all express wnt-2b, wnt-5a, and wnt-11. Amplification products were resolved on 1.5% agarose gels and visualized by eithidium bromide staining. Size markers (base pairs) are shown in the left lane. Two other ovary cancer cell lines (HEY and SW626) provided the same results (not shown). Similar analyses with at least two different RNA preparations from these cell lines provided the same results.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Members of the wnt family have been implicated in a number of developmental processes in both insects and vertebrates. These include the regulation of cell migration, cell proliferation/apoptosis, cell differentiation, as well as tissue patterning. The development and growth of ovarian follicles involves all of these processes. Here, we report that mRNAs corresponding to components of the wnt signaling cascade, wnt-2, wnt-2b, fzd-4, and dsh-2, are expressed in the immature rat ovary. The expression of the wnt and fzd mRNAs is compartmentalized and limited to the follicular granulosa cells (wnt-2, fzd-4) and ovarian surface epithelium (wnt-2b). The compartmentalization of the wnt signaling components is consistent with a role for wnt proteins as local regulators of tissue modeling and patterning in the ovary.

The expression of a number of wnt genes (e.g. wnt-2, -2b, -3, -4, and -5a) has been associated with the development and cellular differentiation of tissues and organs of the male and female reproductive systems (22, 23, 44, 45, 46, 47, 48). Wnt-1 is expressed in postmeiotic round spermatids. An effect on male fertility in mice lacking functional wnt-1 (swaying mice) has not, however, been demonstrated (reviewed in Ref. 49). No wnts have been associated with the mammalian female gamete. Vainio et al. (22) have shown that wnt-4 is crucial in establishing female sexual development. Wnt-4 null mutant mice display defects in Müllerian duct differentiation and in ovarian development. The ovaries of these animals show a depleted stock of oocytes and inappropriate steroidogenesis indicative of ovarian somatic cell differentiation along a male phenotypic pathway. In the majority of these studies, however, wnt expression at early stages of the formation and/or differentiation of reproductive tissues was examined. The expression within adult reproductive tissues has been limited to the examination of accessory sex tissues such as the mammary gland, uterus, and prostate. To our knowledge, studies examining wnt expression in the adult mammalian ovary have not been reported.

The function of wnt-2 is not known. It is a member of the "wnt-1 class" of wnts, i.e. it is capable of transforming C57MG cells and inducing axis duplication in Xenopus embryos, although it is not as potent in these properties as other members of this class (50). Wnt-2 is expressed in the developing pericardium (51), lung (52), and placenta (53). Unlike the majority of wnts, wnt-2 is not expressed in the developing CNS or limb. Adult tissue expression has been demonstrated in the lung (32), endometrium (44), mammary gland (47), and colon (54). Ovarian steroids can modulate wnt-2 expression in the endometrium and mammary gland; compartment switching in the mammary gland has been noted in human breast cancer (45). Targeted disruption of the wnt-2 gene in mice results in placentation defects; surviving animals display no fertility defects (53). These observations indicate that wnt-2 is expressed in a number of adult reproductive tissues and displays hormonal regulation. The knockout studies indicate that wnt-2 may not be essential for follicular function, or that other wnts may substitute for its function.

RT-PCR and ISH analyses indicate that wnt-2 is expressed only in follicular granulosa cells. By ISH, occasional staining was seen in oocytes, but this was not consistent. Theca-interstitial cells showed background staining. The granulosa cells of all growing follicles were positive for wnt-2. Primordial follicles were observed in a few sections, and cells in these follicles also appeared positive (data not shown). We plan to examine wnt-2 expression in cells of the primordial follicle by examining the ovaries of neonatal rats where the population of primordial follicles is abundant and dominant.

The ISH signal for wnt-2 appears to be more intense in the centrally located granulosa cells, i.e. the perioocyte, cumulus, and periantral granulosa cells, raising the possibility of a graded expression pattern for this gene. Assuming the in situ expression pattern conforms to wnt-2 protein expression, the centripetal signal may function as a morphogenetic gradient allowing patterning or establishing the boundaries of particular granulosa phenotypes. There is ample evidence from Drosophila that wnts and other patterning genes (e.g. hedgehog) establish planar polarity and cell differentiation through morphogenic gradients (55). We have preliminary evidence that immunoreactive wnt-2 is secreted by granulosa cells, but we do not know if this holds for granulosa cells at all follicular stages. The secretion of wnt proteins in invertebrates is regulated (56).

The fzd family of serpentine, seven-transmembrane containing receptors was identified recently as putative receptors for wnts (reviewed in Ref. 57). Although the deduced structure of fzd is consistent with it being a G protein-coupled receptor, specific G protein association has yet to be demonstrated. The mammalian fzd family presently has ten members (see wnt gene site). Current thinking indicates that the type of fzd is the determinant of which signal transduction pathway is activated (17). Owing to the lack of specific antibodies for wnts, and the difficulty in generating recombinant bioactive wnt, it has been difficult to associate a specific wnt with a specific fzd. We have preliminary evidence, using an immortalized rat granulosa cell line, that wnt-2 can interact with fzd-4. The pattern of fzd-4 expression in the rat ovary indicates that the granulosa cells of all growing follicles express fzd-4. No obvious difference in the intensity of the hybridization signal is seen in the membrana granulosa. Taking into consideration the possibility of a wnt-2 gradient, as discussed above, this raises the potential for graded wnt-2 signaling in the membrana granulosa.

Because the sequence for rat wnt-2b has not been reported, we attempted to obtain the complete cDNA for this gene. A screen of a rat ovarian cDNA library provided an incomplete sequence for wnt2b. Attempts to obtain additional sequence by 5'-RACE yielded no additional sequence. We are attempting, presently, to obtain additional sequence information by screening a rat genomic library. Recent reports indicate that human wnt-2b can undergo alternative splicing (42), and we have preliminary evidence that similar events may occur in the rat.

The localization of wnt-2b expression in the ovary is intriguing. Expression in the surface epithelium is marked. As far as we can determine, the presence or absence of underlying structures (e.g. follicles at different stages of development) does not influence wnt-2b expression. Wnt-2b expression is associated with chick limb and eye development and with differentiation of embryonic carcinoma cells (58, 59). The association with chick limb and eye lens development indicates that this wnt may be involved in modulating cell movements or cell proliferation, respectively, events that would be of importance in the ovarian surface epithelium during postovulatory wound healing. The implication, however, would be that OSE cells would express a fzd. Our results clearly demonstrate that, if this is the case, it is not fzd-4.

The presence of wnt-2b in the human cancer cell lines, as well as in the rodent OSE, suggest that this wnt may be involved in the development and function of this tissue. The transforming capability of wnts in culture is well established (16, 17). Furthermore, alterations in wnt expression have been described in a variety of human tumors, including those of female reproductive tissues (44, 45). It is interesting that we have detected the common expression of three different wnts in cell lines derived from histologically varied human ovarian carcinomas. The expression pattern for wnt in the normal human OSE has not yet been reported. An analysis of wnt expression in normal and malignant ovarian tissue would shed further light on the involvement of wnts in ovarian carcinogenesis.

In summary, our study represents a first attempt at defining a function and mechanism of action for the wnt family gene products in the mammalian adult ovary. The ovary is an example of a plastic adult tissue in which developmental processes occur throughout reproductive life. It is tempting to speculate that wnts may fulfill a role in regulating the remodeling and patterning processes in the ovary.


   Acknowledgments
 
We thank Dr. Michael Melner (Vanderbilt University, Nashville, TN) for providing the rat ovary cDNA libraries. Ms. Debbie Blake (Department of Obstetrics & Gynecology, McGill University) conducted some of the initial sequencing of the wnt and fzd clones. We would like to acknowledge the assistance of Dr. Beat Suter (Department of Biology, McGill University) in the preparation of the DIG-labeled DNA probes.


   Footnotes
 
A.R. was supported by a fellowship from Schweizerische Stiftung für Medizinisch-Biologische Stipendien. These studies were supported by funds from the Canadian Institutes of Health Research.

1 Present address: Ruhr-Universität Bochum, Medizinische Fakultät, Abteilung für Anatomie und Embryologie, Gebäude MA5/162, Universitätsstrasse 159, D-44780 Bochum, Germany. Back

Abbreviations: CG, Chorionic gonadotropin; DIG, digoxygenin; dsh-2, disheveled-2; eCG, equine CG; fzd-4, frizzled-4; hCG, human CG; HPRT, hypoxanthine phosphoribosyltransferase; ISH, in situ hybridization; nt, nucleotides; ORF, open reading frame; OSE, ovarian surface epithelium; SSC, saline sodium citrate.

Received October 19, 2001.

Accepted for publication March 21, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Gumbiner BM 1996 Cell adhesion: the molecular basis of tissue architecture and morphogenesis. Cell 84:345–357[CrossRef][Medline]
  2. Potter E, Bergwitz C, Brabant G 1999 The cadherin-catenin system: implications for growth and differentiation of endocrine tissues. Endocr Rev 20:207–239[Abstract/Free Full Text]
  3. Rowlands TM, Symonds JM, Farookhi R, Blaschuk OW 2000 Cadherins: crucial regulators of structure and function in reproductive tissues. Rev Reprod 5:53–61[Abstract]
  4. Di Carlo A, De Felici M 2000 A role for E-cadherin in mouse primordial germ cell development. Dev Biol 226:209–219[CrossRef][Medline]
  5. Khan-Dawood F, Yang JO, Ozigi AA, Dawood MY 1996 Immunocytochemical localization and expression of E-cadherin, ß-catenin, and plakoglobin in the baboon (Papio anubis) corpus luteum. Biol Reprod 55:246–253[Abstract]
  6. Kirkup KM, Mallin AM, Bagnell CA 2000 Inhibition of pig granulosa cell adhesion and growth in vitro by immunoneutralization of epithelial cadherin. J Reprod Fertil 120:275–281[Abstract]
  7. Machell NH, Blaschuk OW, Farookhi R 2000 Developmental expression and distribution of N- and E-cadherin in the rat ovary. Biol Reprod 63:797–804[Abstract/Free Full Text]
  8. Mackay S, Nicholson CL, Lewis SP, Brittan M 1999 E-cadherin in the developing mouse gonad. Anat Embryol (Berl) 200:91–102[CrossRef][Medline]
  9. Harandian F, Farookhi R 1998 Contact-dependent cell interactions determine hormone responsiveness and desensitization in rat granulosa cells. Endocrinology 139:1700–1707[Abstract/Free Full Text]
  10. Farookhi R, Blaschuk OW 1989 E-cadherin may be involved in mediating FSH-stimulated responses in rat granulosa cells. In: Hirshfield AN, ed. VII Ovarian workshop: paracrine communication in the ovary: ontogenesis and growth factors. New York: Plenum Press; 257–265
  11. Farookhi R, Blaschuk OW 1991 Cadherins and ovarian follicular development. In: Gibori G, ed. Signalling mechanisms and gene expression in the ovary. New York: Springer Verlag; 254–260
  12. Farookhi R, Desjardins J 1986 Luteinizing hormone receptor induction in dispersed granulosa cells requires estrogen. Mol Cell Endocrinol 47:13–24[CrossRef][Medline]
  13. Peluso JJ, Pappalardo A 1994 Progesterone and cell-cell adhesion interact to regulate rat granulosa cell apoptosis. Biochem Cell Biol 72:547–551[Medline]
  14. Peluso JJ, Papparlardo A, Trolice MP 1996 N-cadherin-mediated cell contact inhibits granulosa cell apoptosis in a progesterone-independent manner. Endocrinology 137:1196–1203[Abstract]
  15. Trolice MP, Pappalardo A, Peluso JJ 1997 Basic fibroblast growth factor and N-cadherin maintain rat granulosa cell and ovarian surface epithelial cell viability by stimulating the tyrosine phosphorylation of the fibroblast growth factor receptors. Endocrinology 138:107–113[Abstract/Free Full Text]
  16. Dale TC 1998 Signal transduction by the Wnt family of ligands. Biochem J 329:209–223
  17. Wodarz A, Nusse R 1998 Mechanisms of Wnt signaling in development. Annu Rev Cell Dev Biol 14:59–88[CrossRef][Medline]
  18. Pandur P, Kuhl M 2001 An arrow for wingless to take-off. Bioessays 23:207–210[CrossRef][Medline]
  19. Kuhl M, Sheldahl LC, Malbon CC, Moon RT 2000 Ca2+/calmodulin-dependent protein kinase II is stimulated by Wnt and Frizzled homologs and promotes ventral cell fates in Xenopus. J Biol Chem 275:12701–12711[Abstract/Free Full Text]
  20. Kuhl M, Sheldahl LC, Park M, Miller JR, Moon RT 2000 The Wnt/Ca2+ pathway: a new vertebrate Wnt signaling pathway takes shape. Trends Genet 16:279–283[CrossRef][Medline]
  21. Rulifson EJ, Wu CH, Nusse R 2000 Pathway specificity by the bifunctional receptor frizzled is determined by affinity for wingless. Mol Cell 6:117–126[CrossRef][Medline]
  22. Vainio S, Heikkila M, Kispert A, Chin N, McMahon AP 1999 Female development in mammals is regulated by Wnt-4 signalling. Nature 397:405–409[CrossRef][Medline]
  23. Parr BA, McMahon AP 1998 Sexually dimorphic development of the mammalian reproductive tract requires Wnt-7a. Nature 395:707–710[CrossRef][Medline]
  24. McClay DW, Clarke HJ 1997 The ability to organize sperm DNA into functional chromatin is acquired during meiotic maturation in murine oocytes. Dev Biol 186:73–84[Medline]
  25. Noubani A, Farookhi R, Gutkowska J 2000 B-type natriuretic peptide receptor expression and activity are hormonally regulated in rat ovarian cells. Endocrinology 141:551–559[Abstract/Free Full Text]
  26. Chomczynski P, Sacchi N 1987 Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159[Medline]
  27. Gavin BJ, McMahon AP 1993 Cloning developmentally regulated gene families. Methods Enzymol 225:653–663[Medline]
  28. Gavin BJ, McMahon JA, McMahon AP 1990 Expression of multiple novel Wnt-1/int-1-related genes during fetal and adult mouse development. Genes Dev 4:2319–2332[Abstract/Free Full Text]
  29. Wang Y, Macke JP, Abella BS, Andreasson K, Worley P, Gilbert DJ, Copeland NG, Jenkins NA, Nathans J 1996 A large family of putative transmembrane receptors homologous to the product of the Drosophila tissue polarity gene frizzled. J Biol Chem 271:4468–4476[Abstract/Free Full Text]
  30. Tanaka S, Akiyoshi T, Mori M, Wands JR, Sugimachi K 1998 A novel frizzled gene identified in human esophageal carcinoma mediates APC/beta-catenin signals. Proc Natl Acad Sci USA 95:10164–10169[Abstract/Free Full Text]
  31. Tsang M, Lijam N, Yang Y, Beier DR, Wynshaw-Boris A, Sussman DJ 1996 Isolation and characterization of mouse dishevelled-3. Dev Dyn 207:253–262[CrossRef][Medline]
  32. Levay-Young BK, Navre M 1992 Growth and developmental regulation of wnt-2 (irp) gene in mesenchymal cells of fetal lung. Am J Physiol 262:L672–L683
  33. Zakin LD, Mazan S, Maury M, Martin N, Guenet JL, Brulet P 1998 Structure and expression of Wnt13, a novel mouse Wnt2 related gene. Mech Dev 73:107–116[CrossRef][Medline]
  34. Murphy E, Hieny S, Sher A, O’Garra A 1993 Detection of in vivo expression of interleukin-10 using a semi-quantitative polymerase chain reaction method in Schistosoma mansoni infected mice. J Immunol Methods 162:211–223[CrossRef][Medline]
  35. Heckert LL, Daley IJ, Griswold MD 1992 Structural organization of the follicle-stimulating hormone receptor gene. Mol Endocrinol 6:70–80[Abstract/Free Full Text]
  36. Akatsuka K, Yoshida-Komiya H, Tulsiani DR, Orgebin-Crist MC, Hiroi M, Araki Y 1998 Rat zona pellucida glycoproteins: molecular cloning and characterization of the three major components. Mol Reprod Dev 51:454–467[CrossRef][Medline]
  37. Bockmann J, Winter C, Kreutz MR, Wittkowski W, Böckers TM 1998 A PCR strategy for screening DNA libraries. Technical Tips Online 1:T01373
  38. Suter B, Steward R 1991 Requirement for phosphorylation and localization of the Bicaudal-D protein in Drosophila oocyte differentiation. Cell 67:917–926[CrossRef][Medline]
  39. Dietl J, Marzusch K 1993 Ovarian surface epithelium and human ovarian cancer. Gynecol Obstet Invest 35:129–135[Medline]
  40. Buick RN, Pullano R, Trent JM 1985 Comparative properties of five human ovarian adenocarcinomal cell lines. Cancer Res 45:3668–3676[Abstract/Free Full Text]
  41. Katoh M, Hirai M, Sugimura T, Terada M 1996 Cloning, expression and chromosomal localization of Wnt-13, a novel member of the Wnt gene family. Oncogene 13:873–876[Medline]
  42. Katoh M, Kirikoshi H, Saitoh T, Sagara N, Koike J 2000 Alternative splicing of the WNT-2B/WNT-13 gene. Biochem Biophys Res Commun 275:209–216[CrossRef][Medline]
  43. Klingensmith J, Yang Y, Axelrod JD, Beier DR, Perrimon N, Sussman DJ 1996 Conservation of dishevelled structure and function between flies and mice: isolation and characterization of Dvl2. Mech Dev 58:15–26[CrossRef][Medline]
  44. Bui TD, Zhang L, Rees MC, Bicknell R, Harris AL 1997 Expression and hormone regulation of Wnt2, 3, 4,5a, 7a, 7b and 10b in normal human endometrium and endometrial carcinoma. Br J Cancer 75:1131–1136[Medline]
  45. Dale T, Weber-Hall S, Smith K, Huguet E, Jayatilake H, Gusterson B, Shuttleworth G, O’Hare M, Harris A 1996 Compartment switching of WNT-2 expression in human breast tumors. Cancer Res 56:4320–4323[Abstract/Free Full Text]
  46. Huguet EL, McMahon JA, McMahon AP, Bicknell R, Harris AL 1994 Differential expression of human Wnt genes 2,3,4, and 7B in human breast cell lines and normal and disease states of human breast tissue. Cancer Res 54:2615–2621[Abstract/Free Full Text]
  47. Weber-Hall SJ, Phippard DJ, Niemeyer CC, Dale TC 1994 Developmental and hormonal regulation of Wnt gene expression in the mouse mammary gland. Differentiation 57:205–214[CrossRef][Medline]
  48. Miller C, Pavlova A, Sassoon DA 1998 Differential expression patterns of Wnt genes in the murine female reproductive tract during development and the estrous cycle. Mech Dev 76:91–99[CrossRef][Medline]
  49. Nusse R, Varmus HE 1992 Wnt genes. Cell 69:1073–1087[CrossRef][Medline]
  50. Smalley MJ, Dale TC 1999 Wnt signalling in mammalian development and cancer. Cancer Metastasis Rev 18:215–230[CrossRef][Medline]
  51. McMahon JA, McMahon AP 1989 Nucleotide sequence, chromosomal localization and developmental expression of the mouse int-1-related gene. Development 107:643–650[Abstract]
  52. Bellusci S, Henderson R, Winnier G, Oikawa T, Hogan BLM 1996 Evidence from normal expression and targeted misexpression that bone morphogenetic protein-4 (Bmp-4) plays a role in mouse embryonic lung morphogenesis. Development 122:1693–1702[Abstract]
  53. Monkley SJ, Delaney SJ, Pennisi DJ, Christiansen JH, Wainwright BJ 1996 Targeted disruption of the Wnt2 gene results in placentation defects. Development 122:3343–3353[Abstract]
  54. Vider BZ, Zimber A, Chastre E, Prevot S, Gespach C, Estlein D, Wolloch Y, Tronick SR, Gazit A, Yaniv A 1996 Evidence for the involvement of the Wnt 2 gene in human colorectal cancer. Oncogene 12:153–158[Medline]
  55. Teleman AA, Strigini M, Cohen SM 2001 Shaping morphogen gradients. Cell 105:559–562[CrossRef][Medline]
  56. Burrus LW, McMahon AP 1995 Biochemical analysis of murine Wnt proteins reveals both shared and distinct properties. Exp Cell Res 220:363–373[CrossRef][Medline]
  57. Orsulic S, Peifer M 1996 Cell-cell signalling: wingless lands at last. Curr Biol 6:1363–1367[CrossRef][Medline]
  58. Wakeman JA, Walsh J, Andrews PW 1998 Human Wnt-13 is developmentally regulated during the differentiation of Ntera-2 pluripotent human embryonal carcinoma cells. Oncogene 17:179–186[CrossRef][Medline]
  59. Kawakami Y, Capdevila J, Buscher D, Itoh T, Esteban CR, Belmonte JC 2001 Wnt signals control FGF-dependent limb initiation and aer induction in the chick embryo. Cell 104:891–900[CrossRef][Medline]



This article has been cited by other articles:


Home page
 Endocr. Rev.Home page
M. A. Edson, A. K. Nagaraja, and M. M. Matzuk
The Mammalian Ovary from Genesis to Revelation
Endocr. Rev., October 1, 2009; 30(6): 624 - 712.
[Abstract] [Full Text] [PDF]

Home page
 ReproductionHome page
E. Nilsson, G. Dole, and M. K Skinner
Neurotrophin NT3 promotes ovarian primordial to primary follicle transition
Reproduction, October 1, 2009; 138(4): 697 - 707.
[Abstract] [Full Text] [PDF]

Home page
 ReproductionHome page
N. Golestaneh, E. Beauchamp, S. Fallen, M. Kokkinaki, A. Uren, and M. Dym
Wnt signaling promotes proliferation and stemness regulation of spermatogonial stem/progenitor cells
Reproduction, July 1, 2009; 138(1): 151 - 162.
[Abstract] [Full Text] [PDF]

Home page
 Mol Hum ReprodHome page
H.-X. Wang, F. R. Tekpetey, and G. M. Kidder
Identification of WNT/{beta}-CATENIN signaling pathway components in human cumulus cells
Mol. Hum. Reprod., January 1, 2009; 15(1): 11 - 17.
[Abstract] [Full Text] [PDF]

Home page
 Hum ReprodHome page
P. A. Fowler, D. R. Abramovich, N. E. Haites, P. Cash, N. P. Groome, A. Al-Qahtani, T. J. Murray, and R. G. Lea
Human fetal testis Leydig cell disruption by exposure to the pesticide dieldrin at low concentrations
Hum. Reprod., November 1, 2007; 22(11): 2919 - 2927.
[Abstract] [Full Text] [PDF]

Home page
 Endocr. Rev.Home page
C. Stocco, C. Telleria, and G. Gibori
The Molecular Control of Corpus Luteum Formation, Function, and Regression
Endocr. Rev., February 1, 2007; 28(1): 117 - 149.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
D. Boerboom, L. D. White, S. Dalle, J. Courty, and J. S. Richards
Dominant-Stable {beta}-Catenin Expression Causes Cell Fate Alterations and Wnt Signaling Antagonist Expression in a Murine Granulosa Cell Tumor Model
Cancer Res., February 15, 2006; 66(4): 1964 - 1973.
[Abstract] [Full Text] [PDF]

Home page
 J. Mol. Diagn.Home page
A. Steg, W. Wang, C. Blanquicett, J. M. Grunda, I. A. Eltoum, K. Wang, D. J. Buchsbaum, S. M. Vickers, S. Russo, R. B. Diasio, et al.
Multiple Gene Expression Analyses in Paraffin-Embedded Tissues by TaqMan Low-Density Array: Application to Hedgehog and Wnt Pathway Analysis in Ovarian Endometrioid Adenocarcinoma
J. Mol. Diagn., February 1, 2006; 8(1): 76 - 83.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
D. Boerboom, M. Paquet, M. Hsieh, J. Liu, S. P. Jamin, R. R. Behringer, J. Sirois, M. M. Taketo, and J. S. Richards
Misregulated Wnt/{beta}-Catenin Signaling Leads to Ovarian Granulosa Cell Tumor Development
Cancer Res., October 15, 2005; 65(20): 9206 - 9215.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Endocrinol.Home page
E. Jansen, J. S. E. Laven, H. B. R. Dommerholt, J. Polman, C. van Rijt, C. van den Hurk, J. Westland, S. Mosselman, and B. C. J. M. Fauser
Abnormal Gene Expression Profiles in Human Ovaries from Polycystic Ovary Syndrome Patients
Mol. Endocrinol., December 1, 2004; 18(12): 3050 - 3063.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
M. Hsieh, S. M. Mulders, R. R. Friis, A. Dharmarajan, and J. S. Richards
Expression and Localization of Secreted Frizzled-Related Protein-4 in the Rodent Ovary: Evidence for Selective Up-Regulation in Luteinized Granulosa Cells
Endocrinology, October 1, 2003; 144(10): 4597 - 4606.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ricken*, A.
Right arrow Articles by Farookhi, R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ricken*, A.
Right arrow Articles by Farookhi, R.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals